Vaccine for intrauterine disease

ABSTRACT

Provided are compositions and methods for use in prophylaxis of puerperal metritis and improving reproductive function of ruminants. The methods and compositions are for subcutaneous administration and are provided as veterinary compositions and as articles of manufacture. The veterinary composition can contain whole cells selected from whole cells of  Escherichia coli  ( E. coli ),  Trueperella pyogenes  ( T. pyogenes ),  Fusobacterium necrophorum  ( F. necrophorum ) and combinations thereof; and/or proteins selected from  F. necrophorum  leukotoxin (LKT),  E. coli  type 1 fimbrial adhesin (FimH),  T. pyogenes  pyolysin (PLO), and all combinations of the whole cells and the proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/731,333, filed on Nov. 29, 2012, the disclosure of which isincorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to health of ruminants and morespecifically to compositions and methods for mitigating the effects ofbacterial infections which are related to uterine disease.

BACKGROUND OF THE DISCLOSURE

Postpartum uterine diseases of dairy cows compromise animal welfare andmay result in early removal from the herd or impaired reproductiveperformance. Puerperal metritis is defined by an abnormally enlargeduterus and a fetid, watery, red-brown uterine discharge associated withsigns of systemic illness (decreased milk yield, dullness, or othersigns of toxemia) and temperature>39.5° C. within 21 d afterparturition, while endometritis refers to inflammation of the uteruswithout systemic illness, happening later than 21 d postpartum. In NorthAmerica, metritis affects 10% to 20% of cows, whereas the incidence ofendometritis is approximately 28%, ranging from 5.3% to 52.6%. Puerperalmetritis is commonly treated with antibiotics like penicillin orthird-generation cephalosporins. However, antibiotic resistanceworldwide is recognized as a top public health challenge and thus thereis growing concern regarding the potential impact of extensive use ofantibiotics in food animals, including later-generation cephalosporins.The cost of each case of metritis has been reported at approximatelyUS$329-386, due to antibiotic treatment and the detrimental effects ofmetritis on reproductive performance, milk production, andsurvivability. Thus, there is an ongoing and unmet need for compositionsand methods for use in prophylaxis against uterine diseases. The presentdisclosure meets these needs.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure provides in one aspect a method for prophylaxisof puerperal metritis in a ruminant. The method generally comprisessubcutaneously administering a veterinary composition to a ruminantmammal. The veterinary composition can comprise whole cells selectedfrom whole cells of Escherichia coli (E. coli), Trueperella pyogenes (Tpyogenes), Fusobacterium necrophorum (F. necrophorum) and combinationsthereof; and/or (2) proteins selected from F. necrophorum leukotoxin(LKT), E. coli type 1 fimbrial adhesin (FimH), T. pyogenes pyolysin(PLO), and all combinations of the whole cells and the proteins. Inembodiments, the veterinary composition comprises the whole cells of E.coli, T. pyogenes, and F. necrophorum. In embodiments, the compositioncomprises the FimH, PLO, and LKT proteins. In embodiments, theveterinary composition comprises the whole cells of E. coli, T.pyogenes, and F. necrophorum and the FimH, PLO, and LKT proteins.

The method is expected to be suitable for use with any ruminant mammal.In embodiments, the ruminant is a member of the genus Bos, such as anox, cow, or buffalo, and it certain embodiments the ruminant is a dairycow.

In one approach, the dairy cow is a member of a group of dairy cows andthe method includes subcutaneously administering the veterinarycomposition to additional dairy cows in the group such that theincidence of puerperal metritis in the group is reduced.

In another aspect the disclosure includes improving reproductivefunction of a ruminant. The method of improving the reproductivefunction of a ruminant comprises subcutaneously administering to theruminant a veterinary composition as described herein such that thereproductive function of the ruminant is improved.

In another aspect the disclosure includes a veterinary compositioncomprising whole cells selected from whole cells of E. coli, T.pyogenes, F. necrophorum and combinations thereof; proteins selectedfrom F. necrophorum LKT, E. coli FimH, PLO, and combinations thereof;and any combination of the whole cells and the proteins.

In another aspect an article of manufacture is provided. The article ofmanufacture comprises packaging and at least one sealed container. Thecontainer contains a veterinary composition comprising whole cellsselected from whole cells of E. coli, T. pyogenes, F. necrophorum andcombinations thereof; proteins selected from F. necrophorum LKT, E. coliFimH, PLO, and combinations thereof; and any combination of the wholecells and the proteins. The packaging further comprises printed materialproviding an indication that the veterinary composition is forsubcutaneous administration to a ruminant for prophylaxis of puerperalmetritis, and/or for increasing the reproductive function of theruminant.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of the prevalence of bacterial species-specificvirulence factors, lktA (Fusobacterium necrophorum), fimH (Escherichiacoli), and fimA and plo (Arcanobacterium pyogenes) at 3 different stagesof lactation. *V.F.≧1=At least one virulence factor present.

FIG. 2 shows an example of an effect of fimH detection at 1-3 days inmilk (DIM) on reproductive performance. The solid black line representsfimH negative cows and the interrupted line fimH positive cows. fimHpositive cows were 2.1 times less likely to be confirmed pregnant thanfimH negative cows (P<0.001).

FIG. 3 shows an example of an effect of vaccination on rectaltemperature at 6±1 DIM. Vaccines were evaluated separately (A,P-value=0.14), and grouped (B, P-value=0.018). Standard errors of themeans are represented by the error bars.

FIG. 4 shows an example of an effect of vaccination on ELISA-detectedserum IgG against E. coli (A), FimH (B), F. necrophorum (C), LKT (D), T.pyogenes (E), and PLO (F). X-axis represents days relative to calving,while Y-axis represents OD₆₅₀ of ELISA-detected serum IgG againstseveral antigens. Standard errors of the means are represented by theerror bars.

FIG. 5 shows an example of Kaplan-Meier survival analysis ofcalving-to-conception interval by treatment grouped as control,subcutaneous vaccines and intravaginal vaccines. The mediancalving-to-conception interval for subcutaneous vaccines (innerinterrupted line), intravaginal vaccines (middle interrupted line), andcontrol (solid line) was 94, 114, and 120 respectively. (P-value=0.04).

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is generally directed to compositions and methodsfor improving the health of ruminants, and more specifically toprophylaxis of intrauterine disease of ruminants.

We evaluated several different approaches for developing compositionsand methods aimed at mitigating intrauterine diseases in ruminants andevaluated these approaches for a variety of effects on metritis andendometritis, among other responses. As is known in the art, metritisgenerally involves inflammation of the wall of the uterus, whileendometritis generally involves inflammation of the endometrium. Ourapproaches included making distinct vaccine formulations and evaluatingthe effects of the different formulations and administration routes onaspects of ruminant health. These effects include stimulation ofimmunological responses against bacteria and bacterial proteins, effectson endometritis and metritis, effects on intrauterine bacterialcontamination, effects on symptoms related to uterine disease, such asrectal temperature, and effects on reproductive function. The results weobtained were unexpected in that we discovered only subcutaneousadministration was prophylactic for puerperal metritis, and onlysubcutaneous administration was effective for improving reproductivefunction in ruminants. We also determined that intravaginal andsubcutaneous vaccination induced a significant increase in serum IgGtiters against all antigens, but subcutaneous vaccination was moreeffective at stimulating IgG production. However, notwithstanding thepositive results for prophylaxis of metritis and improvements inreproductive function obtained using subcutaneous administration, noneof the vaccines, regardless of route of administration, protectedagainst endometritis or significantly decreased the likelihood ofintrauterine bacterial contamination. Thus, as will be more fullydescribed below, the present invention provides novel and effectivecompositions and methods for prophylaxis of puerperal metritis and forimproving reproductive function in ruminants, as well as other benefitsthat will be apparent to those skilled in the art from the presentdisclosure.

In embodiments the ruminant to which a composition of the invention isadministered is a member of the genus Bos, such as an ox, cow, orbuffalo. In one embodiment the ruminant is a dairy cow.

The present disclosure includes compositions comprising whole cellsand/or one or more other immunogens. In embodiments the compositionscomprise whole cells selected from whole cells of Escherichia coli (E.coli), Trueperella pyogenes (T pyogenes), Fusobacterium necrophorum (F.necrophorum) and combinations thereof. In embodiments the compositionscomprise proteins selected from F. necrophorum leukotoxin (LKT), E. colitype 1 fimbrial adhesin (FimH), T. pyogenes pyolysin (PLO), andcombinations thereof. In embodiments the compositions comprise anycombinations of the whole cells and the proteins. Thus, the compositionsmay comprise one, two or three distinct types of bacteria, and/or one,two or three of the proteins. In embodiments, more than one strain ofany bacteria type can be included in the veterinary composition.Additional types of bacteria and proteins can also be included.

Any of the proteins described herein can be isolated from bacteria whichproduce the proteins endogenously and, if desired, purified to anydegree of purity. Any of the proteins can also be reproducedrecombinantly using conventional techniques, such as by expression usingany suitable expression vector and expression system. The amino acidsequences and the polynucleotide sequences encoding the amino acidsequences of each of the proteins described herein are known in the art.

In embodiments, the composition comprises a veterinarily acceptablecarrier, excipient or diluent such that the composition is a veterinarycomposition. Suitable carriers, excipients and diluents are known in theart. In embodiments the veterinary composition functions as a vaccine.

The whole bacterial cells in the veterinary composition can beinactivated using any of a wide variety of methods. Inactivated bacteriaare those that have been treated so that they have less pathogenicityrelative to bacteria that are not similarly treated. In embodiments thebacteria are inactivated such as by exposure to an organic solvent, onenon-limiting example of which is formalin.

The veterinary compositions may also comprise any other agents thatwould be expected to provide a therapeutic and/or prophylactic benefitto the recipient, such as antibiotics and/or adjuvants. Adjuvants thatare suitable for use with veterinary compositions are well known in theart. In one embodiment, the adjuvant is aluminum hydroxide. Non-limitingexamples of other adjuvants include liposomes and archaeosomes, calciumsalts, oil emulsions, nanoparticles and microparticles, saponins,immune-stimulating complexes, nonionic block copolymers, derivatizedpolysaccharides, carrier proteins, bacterial products and theirderivatives, cytokines, complement derivatives, and others. In certainembodiments, the method can be performed prior to, concurrently, orsubsequent to conventional anti-bacterial and anti-inflammatoryapproaches, including but not limited to antibiotic regimens.

In an embodiment, the present disclosure includes an article ofmanufacture comprising packaging and at least one sealed container. Thesealed container can comprise the veterinary compositions. Theveterinary compositions comprise whole cells selected from E. coli, T.pyogenes, F. Necrophorum and combinations thereof. The container cancomprise proteins selected from F. necrophorum LKT, E. coli FimH, T.pyogenes PLO, and combinations thereof. In embodiments the containerincludes any combinations of the whole cells and the proteins. Inembodiments, the packaging can have more than one container whichseparately contains any one or any combination of the cells and/or theproteins. The packaging comprises printed material providing anindication that the veterinary composition is for administration to aruminant and can include a description of subcutaneous administration,and an indication that the administration is for prophylaxis of metritisin a ruminant, and/or to improve the reproductive function of theruminant.

The disclosure provides methods for making veterinary compositions andarticles of manufacture. The method of making a veterinary compositioncomprises providing whole cells of E. coli, and/or T. pyogenes, and/orF. necrophorum, and/or LKT, and/or FimH, and/or PLO, and combining oneor more of these agents with a veterinarily acceptable carrier,excipient and/or diluent to provide a veterinary composition for use inprophylaxis of metritis and/or for use in improving the reproductivefunction of a ruminant. To make an article of manufacture, theveterinary compositions are placed in suitable containers and packaged.The package is made to include printed material which provides anindication that the veterinary compositions are for use in subcutaneousadministration for the prophylaxis of metritis, and/or for improving thereproductive function of ruminants. The printed material can be part ofthe packaging material, or it can be a paper insert, or it can be alabel affixed to, for example, the container(s).

In another aspect the present disclosure includes a method ofstimulating an immune response in a ruminant. The method comprisesadministering a veterinary composition described herein to a ruminant.The immune response can comprise a humoral and/or cell mediatedresponse. The humoral response can comprise an increase inimmunglobulins specific for any antigen expressed by the bacteria,and/or specific for any of the proteins administered in the veterinarycomposition. In embodiments the stimulated immunoglobulins are IgG. Theimmune response in certain embodiments provides a prophylactic effectagainst puerperal metritis. The terms “Prophylaxis” and “prophylactic”as used herein means at least partial inhibition of the formation and/orpersistence of symptoms associated with a uterine disease, and inparticular with puerperal metritis.

Those skilled in the art will be able to determine, given the benefit ofthe present disclosure, when and how frequently to administer thecompositions and how much of each immunogenic agent to include. Ingeneral, factors that will go into this determination include but arenot necessarily limited to the type, size, age and overall health of theruminant. In embodiments, the compositions are administered to theruminant prior to parturition, but can be administered during pregnancyor any time during the life cycle.

In certain embodiments of the present disclosure subcutaneousadministration of a veterinary composition to a ruminant lessens anincrease in rectal temperature of the ruminant due to a bacterialinfection, relative to the rectal temperature of a ruminant with thebacterial infection but to which the veterinary compositions was notadministered.

In an embodiment, administering a veterinary composition describedherein to a ruminant improves the reproductive function of the ruminant.An improvement in reproductive function can comprise a reduction in acalving-to-conception interval relative to the calving-to-conceptioninterval in a ruminant to which a veterinary composition has not beenadministered.

In embodiments the present disclosure includes lowering the incidence ofpuerperal metritis in a group of animals comprising administering aveterinary composition as described herein subcutaneously to members ofa group of animals such that the incidence of puerperal metrtitis in thegroup of animals is lower than if the composition had not beenadministered. In certain embodiments the group of animals is dairy cows.The group of animals can be present in, for example, a dairy farm of anyscale, ranging from a few dairy cows to a commercial dairy farm whichmay house thousands of dairy cows.

The following examples are presented to illustrate the presentdisclosure. They are not intended to limiting in any manner.

EXAMPLE 1

This example shows the relationship between bacterial species-specificvirulence factors (VFs) present in the uterus at 3 different stages oflactation (1-3, 8-10, and 34-36 Days In Milk (DIM)) and the incidence ofmetritis and clinical endometritis in dairy cows. The following VF geneswere investigated: plo (pyolysin), cbpA (collagen-binding protein), andfimA (fimbriae expression) which are Arcanobacterium pyogenes specific;fimH (a type 1 pilus component), Escherichia coli specific; and lktA(leukotoxin), Fusobacterium necrophorum specific. Uterine swabs werecollected from 111 postpartum dairy cows. PCR was used to detect thepresence of plo, cbpA, fimA, fimH, and lktA genes. A. pyogenes cbpA wasdetected in only 5 samples and therefore was not subjected to furtheranalysis. E. coli (fimH) was significantly associated with metritis andendometritis when detected at 1-3 DIM; F. necrophorum (lktA) wassignificantly associated with metritis when detected at 1-3 and 8-12 DIMand with endometritis when detected at 34-36 DIM; and A. pyogenes (fimAand plo) was associated with metritis (fimA) when detected at 1-3 DIMand endometritis (fimA and plo) when detected at 8-10 and 34-36 DIM.

Farm, Management and Sample Collection.

Uterine swabs were collected from 111 post-partum dairy cows that werehoused on a commercial dairy farm located near Ithaca, N.Y. Samples werecollected from April 2010 through June 2010. Reproductive managementutilized a combination of Presynch, Ovsynch, Resynch, and detection ofestrus, with 25 to 30% of cows bred via TAI and the remainder bred afterdetection of estrus solely by activity monitors (ALPRO; DeLaval, KansasCity, Mo.). Uterine secretion samples were collected from each cow threetimes during the study period (at 1-3 DIM, at 8-10 DIM, and at 34-36DIM). Two uterine sample collection methods were used: uterine swab forthe first and second sample and uterine lavage for the third sample. At34-36 DIM the uterus has usually involuted and uterine fluid volume hasdecreased. Thus, at this point, performing uterine lavage is probably abetter way of sampling. Furthermore, uterine lavage was also used at34-36 DIM for the diagnosis of endometritis. Uterine swabs werecollected as follows: cows were restrained and the perineum area wascleansed and disinfected with 70% ethanol. Then, a sterile swab(Har-Vet™ McCullough Double-Guarded Uterine Culture Swab, Spring Valley,Wis.) covered by a sterile pipette (inside a plastic sheath) wasintroduced to the cranial vagina. The pipette was manipulated throughthe cervix into the uterus, the sheath was then ruptured, and the swabwas exposed to uterine secretion. The swab was pulled inside the pipetteand kept in transportation medium at 4° C. until it was processed in thelaboratory. Uterine lavage samples were collected. Briefly, cows wererestrained and the perineum area was cleansed and disinfected with 70%ethanol. A plastic infusion pipette (inside a plastic sheath) wasintroduced to the cranial vagina. The sheath was subsequently ruptured,and the clean pipette tip was manipulated through the cervix into theuterus. A total of 40 ml of sterile saline solution was injected intothe uterus, agitated gently, and a sample of the fluid aspirated. Thevolume of recovered fluid ranged from 5 to 15 ml. Samples were kept inice prior to laboratory processing. This project proposal was reviewedand approved by the Cornell University Institutional Animal Care and UseCommittee (#2011-0111).

Case Definition.

Puerperal metritis was clinically defined as an abnormally enlargeduterus and a fetid, watery, red-brown uterine discharge, with signs ofsystemic illness (decreased milk yield, dullness or other signs oftoxemia) at 8-10 days after parturition and diagnosed by one of theveterinarians of the research team. Clinical endometritis of an obtaineduterine lavage sample was evaluated at 34-36 DIM by visual inspection.In this way we were able to ensure that visible signs of inflammation(purulent or mucupurulent exudate) emanated from the uterus, rather thanfrom another site. All of the uterine lavage samples were visuallyscored by one investigator, who assessed the presence of a purulent ormucopurulent secretion in the uterine lavage sample. The score rangedfrom 0 to 2, with 0 indicating absence of a purulent or mucopurulentsecretion in the lavage sample, 1 indicating a bloody but not purulentsample, and 2 the presence of pus in the lavage sample. Cows with ascore of 2 were considered as diagnosed with clinical endometritis. Bodycondition scores were recorded at the time of each uterine lavage usinga five-point scale with a quarter-point system. Additionally, farmrecorded calving ease score 1-5 (1 and 2 were non-assisted parturitionsand 3-5 were assisted partitions with increasing degree of difficulty),stillbirth parturition, and retained placenta incidence were used asrisk factors.

DNA Extraction, PCR, Gel Electrophoresis and Sequencing.

Swab samples were immersed in 1 ml of phosphate-buffered saline (PBS)into a 15-ml Falcon tube and vortexed to disperse any mucus, bacteria,cells, or transport culture medium. Isolation of total DNA was performedfrom 400 μl of the suspension by using a QIAmp DNA minikit (Qiagen,Santa Clara, Calif.) according to the manufacturer's instructions forDNA purification from blood and body fluids. Some convenientmodifications, such as addition of 400 μg of lysozyme and incubation for12 h at 56° C., were included to maximize bacterial DNA extraction.Total DNA was eluted in 100 μl of sterile DNase/RNase-free water(Promega, Madison, Wis.). DNA concentration and purity were evaluated byoptical density using the Nanodrop ND-1000 spectrophotometer (NanodropTechnologies, Rockland, Del.).

PCR was used for the amplification of specific VFs genes' parts. Amongthe VF genes that contribute to the pathogenic potential of A. pyogenes,three were amplified: plo, cbpA and fimA. To categorize E. coli, thefimH gene was chosen. The leukotoxin gene (lktA), which appears to beunique for F. necrophorum, was used as that bacterium's VF. Detailsregarding the primer sequences, annealing temperatures, and size ofamplicons can be found in Table 1. Presence of known and putative A.pyogenes, E. coli and F. necrophorum VF genes was assessedindependently. Thermal cycling parameters were adjusted according to thetarget sequence, as described in Table 1. All reactions were performedin a 25-μl volume using 24 μl of 1× Green GoTaq Master Mix (made from 2×Green GoTaq Master Mix consisting of Green GoTagReaction Buffer, 400 μMdATP, 400 μM dGTP, 400 μM dCTP, 400 μM dTTP, and 3 mM MgCl₂; PromegaCorp., Madison, Wis.) and primers, and 1 μl of DNA extract. All thermalcycling protocols were performed in a 2720 Thermal Cycler (AppliedBiosystems, Foster City, Calif.). Negative controls consisting of thePCR mixture without DNA were included in all PCR runs. Amplificationproducts were separated by electrophoresis through a 1.2% (wt/vol)agarose gel, stained with 0.5 μg/ml ethidium bromide, and visualizedwith a Kodak Gel Logic 100 Imaging System (GL 100, Scientific ImagingSystems, Eastman Kodak Co., New Haven, Conn.). Positive results wereconsidered to be amplicons of the expected molecular size.

TABLE 1PCR primers and reaction conditions used to amplify virulence factorgene sequences. Annealing Amplicon Target temperature Size genePrimer sequence(5′-3′) (° C.) (bp) ploForward-TCATCAACAATCCACGAAGAG (SEQ ID NO: 1) 60 150Reverse-TTGCCTCCAGTTGACGCTTT (SEQ ID NO: 2) cbpAForward-GCAGGGTTGGTGAAAGAGTTTACT (SEQ ID NO: 3) 60 124Reverse-GCTTGATATAACCTTCAGAATTTGCA (SEQ ID NO: 4) fimAForward-CACTACGCTCACCATTCACAAG (SEQ ID NO: 5) 57 605Reverse-GCTGTAATCCGCTTTGTCTGTG (SEQ ID NO: 6) fimHForward-TGCAGAACGGATAAGCCGTGG (SEQ ID NO: 7) 63 508Reverse-GCAGTCACCTGCCCTCCGGTA (SEQ ID NO: 8) lktAForward-AATCGGAGTAGTAGGTTCTG (SEQ ID NO: 9) 60 401Reverse-TTTGGTAACTGCCACTGC (SEQ ID NO: 10)

To confirm the origin of the PCR products, a random sample of 5 PCRpositive samples from each of the genes fimH, plo, fimA, and lkta waspurified and submitted to sequencing. The sequences obtained werecompared to sequences in GenBank using the BLAST algorithm. Allsequences matched their respective genes with a sequence identity>98%.

Statistical Analysis.

Cows were dichotomized several times into VF positive (1) or VF negative(0). To evaluate the relationship between VF-gene presence and the oddsof metritis and clinical endometritis, two multivariable logisticregression models were fitted to the data; dependent variables weremetritis (yes-no) and clinical endometritis (yes-no). The independentvariables offered to the models were: assisted parturition (calving easescore≧3), retained placenta, parity group (1, 2, ≧3), body conditionscore, and the VFs fimH, plo, fimA, and lktA on DIM 1-3, 8-10, and34-36. All possible two-way interaction terms were added to the model.Variables were manually and stepwise removed from the models indescending order of P-value (backward elimination). Statisticalsignificance was considered when a P<0.05 was observed. Another set ofmultivariable logistic regressions was performed to evaluate theassociation between risk factors (assisted parturition, retainedplacenta, parity, body condition score), and the preceding VFs that weresignificantly associated with uterine disease.

Kaplan-Meier survival analysis was performed to evaluate association ofVF genes (plo, cbpA, fimA, fimH, and lktA) with reproductive performanceusing Medcalc version 10.4.0.0 (Mariakerke, Belgium); the Logrank testwas used to compute P-values. The time series variable for this modelwas the calving-to-conception interval or days from calving until theend of the follow-up period; the minimum follow up period was 150 andthe maximum was 250. Statistical significance was considered when P<0.05was observed.

The following results were obtained using the materials and methodsdescribed above.

Descriptive Statistics.

A total of 111 Holstein cows were used in this study, of which 56(50.4%) were primiparous, 34 (30.6%) were second parity, and 21 (18.9%)were third parity or greater. The A. pyogenes VF cbpA was not identifiedat first sampling (1-3 DIM), and 2 and 3 cows were identified at 8-10,and 34-36 DIM, respectively. Because of its low prevalence VF cbpA wasexcluded from all disease association analyses. The A. pyogenes VFs fimAand plo were prevalent at all 3 different samplings (1-3, 8-10, and34-36 DIM) and the highest prevalence was observed at 8-10 DIM (FIG. 1).The most prevalent VF was the F. necrophorum VF lktA, which encodes anexotoxin (leukotoxin). The E. coli VF fimH was also detected at all 3sampling periods, being most prevalent at 8-10 DIM (FIG. 1). The totalincidence of metritis in this study population was 40.5%, and theprevalence of clinical endometritis diagnosed at 34-36 DIM was 19.8%.

Association of Bacterial Species-Specific Virulence Factors and thePrevalence of Metritis.

Two variables were found to significantly (P<0.05) affect prevalence ofmetritis: E. coli fimH presence at 1-3 DIM and F. necrophorum lktAdiagnosed at 8-10 DIM (Table 2). Cows contaminated with fimH positive E.coli had a 4.7 times higher odds of developing metritis compared to fimHnegative cows (P-value<0.001). F. necrophorum VF lktA was the only VFdetected at 8-10 DIM that was significantly associated with metritis;prevalence of metritis was 54.1% for lktA positive cows and 24% for lktAnegative cows (Table 2).

TABLE 2 Risk factors with a significant effect on the odds of metritisdiagnosed at 8-10 DIM. Metritis Risk factors Number of cows % Odds ratioP-value E. coli fimH 1-3 postpartum POSITIVE 21 76.2% 4.7 <0.01 NEGATIVE90 32.2% Ref. F. necrophorum lktA 8-10 postpartum POSITIVE 61 54.1% 2.60.03 NEGATIVE 50 24.0% Ref.

Association of Bacterial Species-Specific Virulence Factors and thePrevalence of Clinical Endometritis.

Three variables were found to significantly (P<0.05) affect prevalenceof metritis: E. coli fimH presence at 1-3 DIM and A. pyogenes fimAdiagnosed at 8-10 and 34-36 DIM (Table 3). E. coli VF fimH wasassociated with a significantly increased prevalence of endometritisonly when detected in the first sample collection (DIM 1-3); theprevalence of endometritis was 38.1% and 15.6% for fimH positive andnegative cows, respectively (P-value<0.01). Arcanobacterium pyogenesfimA was highly associated with clinical endometritis when detected at8-10 and 34-36 days post-partum; cows that were fimA positive at 34-36DIM were at 8.8 times higher odds of clinical endometritis compared tonegative cows.

TABLE 3 Risk factors with a significant effect on the odds of clinicalendometritis diagnosed at 34-36 DIM Endometritis Risk factors Number ofcows % Odds ratio P-value E. coli fimH1-3 postpartum POSITIVE 21 38.1%5.4 0.01 NEGATIVE 90 15.6% Ref. A. pyogenes fimA 8-10 postpartumPOSITIVE 39 33.3% 5.6 <0.01 NEGATIVE 72 12.5% Ref. A. pyogenes fimA34-36 postpartum POSITIVE 13 61.4% 8.8 <0.01 NEGATIVE 98 14.3% Ref.

Risk Factors for fimH at 1-3 DIM, fimA at 8-10 and 34-36 DIM, and lktAat 8-10 DIM.

Results regarding risk factors with a significant effect on prevalenceof specific VFs (fimH at 1-3 DIM, fimA at 8-10 and 34-36 DIM, and lktAat 8-10 DIM) are presented in Table 4. Cows with a retained placentawere at a 44.8 times higher odds of being contaminated with fimHpositive E. coli compared to cows without a retained placenta and thiswas the stronger effect observed.

TABLE 4 Outcomes of 4 different multivariable logistic regression modelsthat evaluated the association of several risk factors with the odds ofprevalence of the virulence factors fimH (1-3 DIM), fimA (8-10 DIM),fimA (34-36 DIM), and lktA (8-10 DIM). Risk factors n % Odds ratioP-value fimH positive 1-3 DIM Retained placenta Yes 10 90.0% 44.8  <0.01NO 101 11.9% Ref. F. necrophorum lktA 1-3 postpartum POSITIVE 27 44.4%4.2 0.02 NEGATIVE 84 10.7% Ref. fimA positive 8-10 DIM F. necrophorumlktA 1-3 postpartum POSITIVE 27 11.1%  0.12 <0.01 NEGATIVE 84 42.9% Ref.F. necrophorum lktA8-10 postpartum POSITIVE 61 42.6% 2.9 0.01 NEGATIVE50 26.0% Ref. fimA positive 34-36 DIM Ease of parturition Assisted 1040.0 7.2 0.03 Non-assisted 101 8.9 Ref. F. necrophorum lktA 1-3postpartum POSITIVE 27 29.6% 5.8 0.02 NEGATIVE 84 5.9% Ref. F.necrophorum lktA34-36 postpartum POSITIVE 14 57.1% 18.8  <0.01 NEGATIVE97 5.1% Ref. lktA positive 8-10 DIM Ease of parturition Assisted 1090.0% 12.6  0.02 Non-assisted 101 51.5% Ref. E. coli fimH 1-3 postpartumPOSITIVE 21 90.5% 16.2  <0.01 NEGATIVE 90 46.7% Ref

Association of Bacterial Species-Specific Virulence Factors withReproductive Performance.

The only VF significantly associated with reproductive performance wasfimH at 1-3 DIM; cows that were fimH positive at the first sampling were2.1 times less likely to be confirmed pregnant when compared to fimHnegative cows (P<0.01, FIG. 2). As will be apparent from the foregoing,in this example, presence of fimH-carrying E. coli at 1-3 DIM wasstrongly associated with metritis and clinical endometritis. However, E.coli was not associated with uterine diseases when detected at laterstages of lactation (8-10 and 34-36 DIM), suggesting that E. coli islikely among the first bacteria to colonize the intrauterineenvironment, potentially inducing changes that will favor futurecolonization by strict (F. necrophorum) and facultative (A. pyogenes)anaerobic bacteria which will ultimately cause clinical signs of uterinedisease. Intrauterine pathogenic E. coli (IUPEC) possesses an arsenal ofVFs (fimH, hlyA, cdt, kpsMII, ibeA, and astA) associated withextraintestinal pathogenic E. coli (ExPEC). Of the six VFs associatedwith metritis and endometritis, fimH was the most significant because ofits high prevalence and strong association with uterine diseases andreproductive failure.

FimH is an adhesin (from type-1 pilus) that belongs to a family ofproteins involved in bacterial adherence to various targets, includinghost mammalian cells. Results from one previous study showed that clonalE. coli isolated from metritic cows were more adherent to and invasiveinto endometrial epithelial and stromal cells than were clonal bacteriaisolated from clinically unaffected animals. Another study alsoconcluded that the bacteria lacked pathogenicity genes typicallyassociated with virulence in E. coli—they evaluated 17 VF genes and nonewere found to be associated with uterine disease.

In the this example, FimH positive cows at 1-3 DIM were 16.2 times morelikely to develop F. necrophorum intrauterine contamination at 8-10 DIM.These findings support the notion that establishment and persistence ofuterine infection by F. necrophorum and other gram-negative anaerobicbacteria are influenced by the presence of a suitable intrauterineenvironment established by a preceding E. coli infection. Furthermore,in the present study, the presence of fimH-carrying E. coli at 8-10 or34-36 DIM was not associated with metritis, clinical endometritis orreproductive failure. In fact, fimH positive cows at 8-10 DIM had anumerically lower incidence of metritis (32.1%) when compared to fimHnegative cows (43.4%). This fact highlights the multifactorial etiologyof postpartum uterine diseases and the hypothesis that the intrauterinemicrobial population shifts as cows advance into their lactation. Over90% of the fimH positive cows at 1-3 DIM were contaminated with F.necrophorum one week later and the presence of F. necrophorum was animportant risk factor for the appearance of A. pyogenes.

Samples collected at 8-10 DIM presented the highest bacterial prevalencecompared to the other two stages of lactation—at least one VF waspresent in 71% of cows. Interestingly, A. pyogenes and F. necrophorumwere found to predominate in the samples from the second collectionperiod. At 8-10 DIM the VF lktA was the only VF strongly associated withmetritis: the prevalence of metritis was 54.1% for F. necrophorumpositive cows and 24% for the negative cows.

In this example, uterine contamination by F. necrophorum and A. pyogeneswas found to be highest in the second and third sampling periods,respectively, in cows that needed assistance during parturition. Both A.pyogenes and F. necrophorum were strongly associated with clinicalendometritis when detected at the third sampling period (DIM 34-36) inthe study in this example.

In this example, retained placenta increased the odds of intrauterinecontamination by fimH positive E. coli by 44.8 times. Additionally, cowsthat had assisted parturition were at higher risk of F. necrophorum andA. pyogenes contamination. Hence, uterine disease is influenced byparturition events and environmental bacterial contamination plays animportant role.

This example provides evidence that the bacterial etiology of postpartumuterine diseases is dynamic and multifactorial, with a significantcontribution from at least three different bacteria: Escherichia coli,Fusobacterium necrophorum, and Arcanobacterium pyogenes.

Finally, cows that were fimH positive on the first sampling (DIM 1-3)were 2.1 times less likely to become pregnant compared to fimH negativecows; fimH was the only VF gene significantly associated with reducedreproductive performance.

The E. coli specific fimH and the F. necrophorum specific lktA VF geneswere significantly associated with a higher prevalence of metritis whendetected at 1-3 and 8-10 DIM, respectively. The E. coli specific fimHand the A. pyogenes specific fimA VF genes were significantly associatedwith clinical endometritis when detected at 1-3 days (fimH) and at 8-10and 34-36 (fimA) DIM.

EXAMPLE 2

In this example, the efficacy of five vaccine formulations containingdifferent combinations of proteins (FimH; leukotoxin, LKT; and pyolysin,PLO) and/or inactivated whole cells (Escherichia coli, Fusobacteriumnecrophorum, and Trueperella pyogenes) for prophylaxis of postpartumuterine diseases was studied. Our initial expectation was that prepartumimmunization against relevant antigens for postpartum uterine diseaseswould prevent the occurrence of puerperal metritis and endometritis.

As described further below, inactivated whole cells were produced usingtwo genetically distinct strains of each bacterial species (E. coli, F.necrophorum, and T. pyogenes). FimH and PLO subunits were produced usingrecombinant protein expression, and LKT was recovered from culturing awild F. necrophorum strain. Three subcutaneous vaccines were formulated:Vaccine 1 was composed of inactivated bacterial whole cells andproteins; Vaccine 2 was composed of proteins only; and Vaccine 3 wascomposed of inactivated bacterial whole cells only. Two intravaginalvaccines were formulated: Vaccine 4 was composed of inactivatedbacterial whole cells and proteins; and Vaccine 5 was composed of PLOand LKT. To evaluate vaccine efficacy, a randomized clinical trial wasconducted at a commercial dairy farm; 371 spring heifers were allocatedrandomly into one of six different treatments groups: control, Vaccine1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifersassigned to one of the vaccine groups were each vaccinated twice: at 230days and 260 days of pregnancy. When the vaccines were evaluated andgrouped as subcutaneous and intravaginal, the subcutaneous ones werefound to significantly reduce the incidence of puerperal metritis.Additionally, subcutaneous vaccination significantly reduced rectaltemperature at 6±1 days in milk. Reproductive performance was improvedfor cows that received subcutaneous vaccines. In general, vaccinationinduced a significant increase in serum IgG titers against all antigens,with subcutaneous vaccination again being more effective. In conclusion,subcutaneous vaccination with inactivated bacterial components and/orprotein subunits of E. coli, F. necrophorum and T. pyogenes can preventsuccessfully puerperal metritis during the first lactation of dairycows, leading to improved reproductive performance.

Materials and Methods. Inactivated bacterial components. E. coli strains4612-2 and 12714-2 were used for this Example. Strains were grownaerobically on Luria-Bertani (LB) broth (Sigma-Aldrich) at 37° C. Theywere inoculated with 1% of an overnight culture and grown in 800 ml ofmedium, with agitation (150 rpm). For strain 12714-2, cells wereharvested at 4 h, with an OD₆₀₀ of 0.432 and 1.0×10⁹ CFU/ml; for strain4612-2, cells were harvested at 3.5 h, OD₆₀₀ of 0.473 and 1.2×10⁹CFU/ml. The cultures were inactivated with 0.1% formalin for 12 h, andthe cells were concentrated 4-fold (final volume of 200 ml), so 0.25 mlof each strain would be present in the final vaccine formulation, withapproximately 10⁹ CFU per dose.

Trueperella pyogenes strains 10481-8 and 6375-1 were isolated from theuterine lumen of dairy cows. Strains were grown on VersaTREK REDOX 1(Trek Diagnostic Systems, OH) in 7% CO₂ at 37° C. Cells were harvestedat 48 h, with 1.3×10⁸ and 0.5×10⁸ CFU/ml for strains 10481-8 and 6375-1,respectively. The cultures were inactivated with 0.1% formalin for 12 h,and 1 ml of each strain was added to the final vaccine formulation, withapproximately 10⁸ CFU per dose.

Fusobacterium necrophorum strains 5663 and 513 were isolated from theuterine lumen of dairy cows. Strains were grown on VersaTREK REDOX 2(Trek Diagnostic Systems, OH) anaerobically at 37° C. All cultures wereinactivated with 0.1% formalin for 12 h before the cells wereconcentrated. Cells were harvested at 12 h, with 1.6×10¹² and 1.8×10¹²CFU/ml for strains 513 and 5663, respectively. The cultures wereinactivated with 0.1% formalin for 12 h, and 0.01 ml of each strain wasadded to the final vaccine formulation, with approximately 10¹⁰ CFU perdose.

Recombinant protein expression and purification. Bacterial strain growthand induction conditions. E. coli TOP10 (Invitrogen, NY) was growneither on LB agar or in LB broth (Sigma-Aldrich, MO) at 37° C.Ampicillin (50 μg/ml) was added as appropriate. T. pyogenes 49698(American Type Culture Collection, VA) was grown on brain heart infusion(BHI) agar or in BHI broth (BD BBL, MD) supplemented with 5%defibrinated horse blood at 37° C. and 7% CO₂.

For the preparation of His-tagged proteins (His-PLO or FimH₁₋₁₅₆-His),appropriate E. coli cultures were grown at 37° C. with agitation (200rpm) to an optical density at 600 nm of ˜0.6. At this point, isopropyl1-thio-β-D-galactopyranoside (IPTG; Sigma) was added to the cultures to1 mM, which were further incubated with agitation for at least 3 h.

DNA Manipulation and Constructs.

Standard procedures for E. coli transformation and plasmid extraction,DNA restriction, ligation, and agarose gel electrophoresis wereperformed. Primers were synthesized by IDT, and PCRs were performed in aGeneAmp PCR System 9700 (Applied Biosystems, CA). To confirm that nomutations were introduced by PCR, all DNA constructs were sequencedusing an automated DNA sequencer (Cornell Biotechnology Resource Center,NY) and analyzed using LaserGene software (DNASTAR, WI).

Cloning and Purification of Recombinant His-PLO.

The PLO gene, lacking the coding region for the predicted signalsequence, was amplified from A. pyogenes ATCC49698 genomic DNA by PCRwith a 5′ primer containing an XhoI site(5′-ACAGCATCCTCGAGTGCCGGATTGGGAAAC-3′ (SEQ ID NO:11)) and a 3′ primercontaining an EcoRI site (5′-TGGAATTCCCTAGGATTTGACATTGT-3′ (SEQ IDNO:12)). The 50-μl reaction contained 1×Pfx amplification buffer(Invitrogen) with 1 mM MgSO₄ (Invitrogen, NY), 0.3 mM of each dNTP, 0.3μM of each primer, 1 U of Platinum Pfx DNA polymerase (Invitrogen, NY),and approximately 50 ng of template DNA. The cycling parameters foramplification were: initial denaturation for 5 mM at 94° C., followed by30 cycles of denaturation (94° C. for 1 min), annealing (58° C. for 1min), extension (72° C. for 3 min), and a final extension at 72° C. for7 min. The 1.5-kb amplicon was digested with XhoI-EcoRI and cloned intoXhoI-EcoRI-digested pTrcHisB (Invitrogen, NY).

After 3 h of induction, the cells were harvested by centrifugation at10,000×g for 10 min and the pellet was resuspended in 1× Extraction/WashBuffer (50 mM sodium phosphate, 300 mM NaCl) (pH 7.0). Lysozyme wasadded to a final concentration of 0.75 mg/ml and the mixture wasincubated at 4° C. with shaking for 30 min. The cells were disrupted bytwo passages through a French pressure cell (Amicon) at 20,000 psi (138Mpa), and the insoluble material was removed by centrifugation at12,000×g for 30 min. His-PLO was purified from the soluble fraction withTALON metal affinity resin (Clontech, CA) according to themanufacturer's instructions. Isolated pure protein fraction wasconcentrated using a fiber concentration/desalting system using a filterwith a molecular weight exclusion of 10 kDa (Amicon ultra 100K,Millipore, Mass.) and subjected to SDS-PAGE (15%) using the Mini-PROTEANTetra Cell electrophoresis system (Bio-Rad, CA), following standardprotocols. Protein concentration was determined by the Bradford method.

A total of 30 liters of culture was grown to produce a total of 321.24mg of His-PLO. The final volume of His-PLO was 41 ml and the finalconcentration was 7.83 mg/ml.

Cloning and Purification of Recombinant FimH₁₋₁₅₆-his.

The portion of the FimH gene encoding the signal peptide and the first156 amino acids (the mannose-binding lectin domain, LD) of the matureprotein was amplified from plasmid pET-22b(+)-F3-LD, provided by Dr.Evgeni Sokurenko, University of Washington, Wash. The 5′ primer usedcontained a BamHI site (5′-CGCGGATCCATGAAACGTGTTATTACCCTG-3′ (SEQ IDNO:13)) and the 3′ primer contained a HindIII site(5′-CCCAAGCTTCTAGTGATGGTGATGGTGATGGCCGCCAGTAGGCACCAC-3′ (SEQ ID NO:14))and a six-histidine tag following the authentic sequence of the protein.The PCR components were as described for PLO gene amplification. Thecycling parameters for amplification were: initial denaturation for 5min at 94° C., followed by 25 cycles of denaturation (94° C. for 1 min),annealing (61° C. for 1 min), extension (72° C. for 3 min), and a finalextension at 72° C. for 7 min. The amplicon, approximately 0.6 kb, wasdigested with BamHI-HindIII and cloned into BamHI-HindIII-digestedpTrcHisA (Invitrogen). After 5 h of induction, FimH₁₋₁₅₆-Hispurification was performed as described for PLO.

A total of 92 liters of culture was grown to produce 216.34 mg ofFimH₁₋₁₅₆-His. The final volume of FimH₁₋₁₅₆-His was 172.5 ml and theconcentration was 1.25 mg/ml.

Culture Concentrated Supernatant and Affinity Purification ofLeukotoxin.

F. necrophorum strain 6586 was grown in VersaTREK REDOX 2 for 12 hanaerobically at 37° C. The culture supernatant was concentrated at 4°C. in a hollow fiber concentration/desalting system using a filter witha molecular weight exclusion of 100 kDa (Amicon ultra 100K, Millipore,Mass.). Affinity purification of LKT was performed to evaluate theconcentration of LKT in the F. necrophorum 6586 culture concentratedsupernatant. Briefly, purified mAb F7B10 (3.5 mg) was coupled to 5 ml ofAffi-Gel 10 affinity support (Bio-Rad, CA) and packed in a 1×20 cmcolumn. The F. necrophorum 6586 culture concentrated supernatant wasapplied to the column, and non-binding materials were removed by passing15 mL of 0.5 M NaCl in PBS through the column Purified LKT was elutedwith 0.2 M glycine-HCl (pH 3.0), immediately neutralized with NaOH, andwashed and concentrated using an Amicon ultra 10K. Purity of the toxinwas determined by SDS-PAGE.

A total of 10 L of F. necrophorum 6586 was grown to produce 220 mL ofconcentrated supernatant containing 0.186 mg/ml of LKT. The presence andconcentration of LKT in the concentrated supernatant was determined byaffinity purification.

Vaccine formulation. Five different vaccine formulations were made:three subcutaneous vaccines (Vaccines 1-3) and two intravaginal vaccines(Vaccine 4-5). Vaccine 1 was composed of inactivated bacterial wholecells (E. coli, T. pyogenes and F. necrophorum) and proteins (FimH, PLOand LKT); Vaccine 2 was composed only of proteins (FimH, PLO and LKT);and Vaccine 3 was composed only of inactivated bacterial whole cells (E.coli, T. pyogenes and F. necrophorum). Vaccine 4 was composed ofinactivated bacterial whole cells (E. coli, T. pyogenes and F.necrophorum) and proteins (FimH, PLO and LKT), and Vaccine 5 wascomposed only of proteins (PLO and LKT). The adjuvant for thesubcutaneous vaccines was aluminum hydroxide (Rehydragel HPA, GeneralChemical, NJ). The adjuvant volume used in the subcutaneous vaccines was25% of the final vaccine volume. Aluminum hydroxide was added to eachcomponent separately, and it was gently stirred overnight. The adjuvantfor the intravaginal vaccines was 20 μg/dose of Cholera toxin (ListBiological Laboratories, Inc., CA).

All vaccine components were tested for sterility before the finalvaccine was assembled and bottled. Sterility was evaluated by culturing100 μl of vaccine component aerobically in LB broth, aerobically in 7%CO₂ on VersaTREK REDOX 1 and anaerobically on VersaTREK REDOX 2 at 37°C. for 48 h. Components were considered contaminated if there wasbacterial growth in any of the three culture media by the end of theincubation period.

Assessment of endotoxin levels was performed using the LAL EndpointAssay (Hycult Biotech, The Netherlands) following the manufacturer'sinstructions. All vaccine formulations had endotoxin levels below 10⁵EU/ml.

Farm and management. The field trial was conducted in a commercial dairyfarm located near Ithaca, N.Y. Cows were enrolled from May 24, 2012 toAug. 16, 2012; the follow-up period continued until Apr. 30, 2013. Thisfarm was selected because of its long working relationship with theAmbulatory and Production Medicine Clinic at Cornell University. Theresearch protocol was reviewed and approved by the Institutional AnimalCare and Use Committee of Cornell University (Protocol number:2011-0111). The farm milked 3,300 Holstein cows 3 times daily in adouble 52-stall parallel milking parlor. The cows were housed infreestall barns with concrete stalls covered with mattresses and beddedwith manure solids. All cows were offered a total mixed ration (TMR)consisting of approximately 55% forage (corn silage, haylage, and wheatstraw) and 45% concentrate (corn meal, soybean meal, canola, cottonseed,and citrus pulp) on a dry matter basis of the diet. The diet wasformulated to meet or exceed the NRC nutrient requirements for lactatingHolstein cows weighing 650 kg and producing 45 kg of 3.5% fat correctedmilk. The reproductive management utilized a combination of Presynch,Ovsynch, Resynch, and detection of estrus, with 25% to 30% of cows bredvia timed artificial insemination and the remainder bred after detectionof estrus solely by activity monitors (ALPRO; DeLaval, Kansas City,Mo.).

Treatment groups and Case definition. Late pregnant heifers wereenrolled on a weekly basis; inclusion criteria for enrollment were:230±3 days of pregnancy, 629 to 734 days of age and body condition score(BCS) greater than 2.5. Heifers that were visually lame were notincluded in the study. A total randomized field trial study design wasused; heifers were randomly allocated into one of six differenttreatment groups using the random number function of Excel (Microsoft,Redmond, Mass.). A total of 371 pregnant heifers were enrolled in thestudy; 105, 54, 54, 53, 53 and 53 heifers were randomly allocated to thecontrol, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5groups, respectively. Heifers assigned to the vaccine groups receivedtwo doses of vaccine: at 230±3 days of pregnancy and 260±3 days ofpregnancy.

Body condition scores were determined for all study cows at 230±3 daysof gestation, 260±3 days of gestation, 2±1 days in milk (DIM), 6±1 DIMand at 35±3 DIM by a single investigator blinded to treatment groupusing a five-point scale with a quarter-point system. To obtain serumsamples, blood was collected from a coccygeal vein/artery using aVacutainer tube without anticoagulant and a 20 gauge×2.54 cm Vacutainerneedle (Becton, Dickinson and Company, Franklin Lakes, N.J.). All bloodsamples were transported to the laboratory on ice and spun in acentrifuge at 2,000×g for 15 min at 4° C.; serum was harvested andfrozen at −80° C. Serum samples were collected at 230±3 days ofgestation, 260±3 days of gestation, 1±2 DIM, 6±1 DIM and 35±3 DIM.

Cervical swabs were collected at 2±1 DIM and 6±1 DIM; cows wererestrained and the perineum area was cleansed and disinfected with 70%ethanol solution. The swab was manipulated inside the cervix and exposedto uterine secretion. The swabs were kept inside a sterile vial at 4° C.until processed in the laboratory. Swabs collected at 2±1 DIM werecultured aerobically on Chromagar (Difco) at 37° C. and E. coli colonieswere distinguished by a blue color; swabs collected at 6±1 DIM werecultured anaerobically on LKV agar (Anaerobe Systems) and F. necrophorumcolonies were distinguished by morphology.

Retained placenta, puerperal metritis, ketosis, and clinical mastitiswere diagnosed and treated by trained farm personnel who followed aspecific diagnostic protocol designed by veterinarians from theAmbulatory and Production Medicine Clinic, Cornell University. Farmpersonnel were blinded to the treatments.

After parturition, cows were kept in the same pen until around 20 DIM.This pen was monitored intensively by farm employees, and cows weresubmitted to a complete physical exam if they were showing signs ofdullness and depression; cows with fetid, watery, red-brown uterinedischarge accompanied with fever were diagnosed with puerperal metritisand treated by farm employees. Retained placenta was defined as acondition where cows failed to release their fetal membranes within 24 hof calving. Puerperal metritis diagnosis by the research team wasperformed at 6±1 DIM. Puerperal metritis was defined as the presence offetid, watery, red-brown uterine discharge and rectal temperaturegreater than 39.5° C. Information regarding puerperal metritis diagnosiswas not exchanged between farm personnel and the research team. Dataregarding health traits and reproduction were extracted from the farm'sDairyComp 305® database (Valley Agricultural Software, Tulare, Calif.).

Clinical endometritis diagnosis was evaluated at 35±3 DIM by visualinspection of a uterine lavage sample for the presence of purulentsecretion. To obtain uterine lavage samples, the cows were restrained,the perineum area was cleansed and disinfected with 70% ethanol, and aplastic infusion pipette was introduced into the cranial vagina andmanipulated through the cervix into the uterus. A total of 20 ml ofsterile saline solution was infused into the uterus and agitated gently,and a sample of the fluid was aspirated. The volume of recovered fluidranged from 5 to 15 ml. All samples were visually scored by oneinvestigator, who assessed the presence of a purulent or mucopurulentsecretion in the uterine lavage sample. The score ranged from 0 to 2,with 0 indicating absence of a purulent or mucopurulent secretion, 1indicating a bloody but not purulent sample, and 2 indicating thepresence of pus in the lavage sample. Cows with a score of 2 wereconsidered as diagnosed with clinical endometritis. Samples were kept onice until they were cultured on Mueller-Hinton agar plates (BBL™)supplemented with 5% defibrinated sheep blood for 48 h aerobically in 5%CO₂ at 38° C. Typical T. pyogenes colonies were distinguished by colonymorphology, post-incubation hemolysis, and characteristic appearance onGram's stain.

Enzyme-linked immunosorbant assays (ELISAs). Portions of the antigensproduced for preparation of vaccines were used in ELISAs. E. colistrains were pooled together as a single antigen. The same was done forF. necrophorum and T. pyogenes strains.

Bovine serum samples were thawed and mixed before analysis. The selectedELISA protocols were as follows. ELISA micro-titer plates (GreinerBio-One, Germany) were coated with PBS (Phosphate-Buffered Saline 10×,pH 7.4, Ambion®) containing either 0.295 μg/ml of FimH₁₋₁₅₆-His, 0.036μg/ml of His-PLO, 0.186 μg/ml of LKT, 10⁷ cells/ml of E. coli, 10¹⁰cells/ml of F. necrophorum, and 10⁷ cells/ml of T. pyogenes foranti-FimH, anti-LKT, anti-PLO, anti-E. coli, anti-F. necrophorum, andanti-T. pyogenes IgG assays, respectively. Binding of antigen tomicrotiter wells was carried out overnight at 4° C., non-specificbinding sites were blocked with PBS containing 1% casein (ThermoScientific, Rockford, Ill.). Dilutions of bovine serum samples were thenadded to the ELISA plates; serum samples were diluted in proportions of1:1000, 1:5000, 1:5000, 1:150, 1:500, and 1:150 for anti-FimH, anti-LKT,anti-PLO, anti-E. coli, anti-F. necrophorum, and anti-T. pyogenes IgGassays, respectively. The optimal antigen and antibody concentrationswere determined by performing the quantitative ELISA protocol withvarying concentrations. The serotype-specific antibody bound to theELISA plate was detected with anti-bovine IgG antibody conjugated withhorseradish peroxidase, diluted according to the manufacturer'sinstructions (Sigma Aldrich, St. Louis, Mo.), followed by addition ofthe substrate, 3,3′,5,5′-tetramethylbenzidine—TMB (Sigma Aldrich, St.Louis, Mo.). The optical density of each well was measured after 20 minat 650 nm using an ELISA plate reader (Synergy HTmicroplate readerBioTek Instruments, VT). The amount of color produced was proportionalto the amount of primary antibody bound to the proteins on the bottom ofthe wells. Between each step of the assay, the microtiter wells wereaspirated and rinsed 3 times with washing solution (1× PhosphateBuffered Saline Tween-20).

Statistical analyses. Descriptive statistics analysis was undertaken inSAS using the FREQ procedure (SAS Institute INC., Cary, N.C.). To assessthe effect of vaccination on the odds of RDPMET, FDPMET, endometritis,E. coli, F. necrophorum, and T. pyogenes culture outcomes, logisticregression models were fitted in SAS using the Logistic procedure. Theeffect of subcutaneous and intravaginal vaccines on reproduction wasanalyzed by Cox's proportional hazard using the proportional hazardregression procedure in SAS. To assess the effect of vaccination onrectal temperature at 6±1 DIM, mixed general linear models were fittedto the data using JMP®PRO9. To assess the effect of vaccination on ELISAdetecting serum IgG against vaccine antigens, mixed general linearmodels were fitted to the data using JMP®PRO9. For all models describedabove, independent variables and their respective interactions were keptwhen P<0.10 in an attempt to reduce the type II error risk whilemaintaining a stringent type I error risk of 5%. The variable treatmentwas forced into all statistical models even in the absence ofstatistical significance. Age in days at enrollment, and BCS atenrollment were offered to all models.

Using the materials and methods described above in this example, thefollowing results were obtained.

Descriptive statistics. Descriptive statistics regarding average age atenrollment (days), average BCS at enrollment and at 6±1 days postpartum,average gestation length at enrollment, and total number of animalsenrolled are presented in Table 5. Only pregnant heifers were enrolledin this study, allowing us to have as little variation between animalsas possible.

TABLE 5 Descriptive statistics of treatment groups Control Vaccine 1Vaccine 2 Vaccine 3 Vaccine 4 Vaccine 5 Average age (days) at 664 655665 669 666 668 enrollment (±SE) (3.72) (5.2) (5.24) (5.24) (5.24)(5.24) Average body condition 3.71 3.76 3.74 3.65 3.72 3.66 score atenrollment (0.03) (0.05) (0.05) (0.05) (0.05) (0.05) (±SE) Average bodycondition 3.5 3.49 3.52 3.49 3.44 3.50 score at 6 ± 1 (±SE) (0.02)(0.03) (0.03) (0.03) (0.03) (0.03) Average days of 230 230 230 230 230230 gestation at enrollment (0.21) (0.29) (0.29) (0.29) (0.29) (0.29)(±SE) Total enrolled animals 105 54 53 53 53 53

Effect of vaccination on incidence of researcher diagnosed puerperalmetritis (RDPMET), farm diagnosed puerperal metritis (FDPMET), andrectal temperature at 6±1 DIM. The effect of vaccination on theincidence of RDPMET is presented in Table 6. When evaluated separately,vaccines 1, 2 and 3 were associated with numerical reductions in theincidence of RDPMET, whereas vaccines 4 and 5 were associated withincreased incidences. However, these differences were not statisticallysignificant (P-value=0.21). When vaccines were evaluated grouped aseither subcutaneous or intravaginal vaccines, the subcutaneous vaccineswere associated with a significant reduction in the incidence of RDPMET(P-value=0.03).

TABLE 6 Effects of different vaccine formulations on incidence ofresearcher diagnosed puerperal metritis. Vaccines were evaluatedseparately in Model 1, and grouped in Model 2. Age in days and bodycondition score at enrollment were offered to both models Puerperalmetritis Model and incidence Coefficients variables (%) (SE) Odds ratio(95% CI) P-value Model 1 Control 12.12 Ref. baseline 0.21 Vaccine 1 6.25−0.14 (0.55) 0.48 (0.13-1.80) Vaccine 2 4.08 −0.59 (0.64) 0.31(0.07-1.44) Vaccine 3 2.04 −1.30 (0.86) 0.15 (0.02-1.20) Vaccine 4 13.46  0.70 (0.42) 1.13 (0.41-3.01) Vaccine 5 14.00   0.75 (0.42) 1.18(0.43-3.21) Intercept −2.56 (0.25) Model 2 Control 12.12 Ref. baseline0.03 Subcutaneous 4.11 −0.83 (0.31) 0.31 (0.11-0.86) Intravaginal 13.73  0.48 (0.26) 1.15 (0.50-2.63) Intercept −2.32 (0.20)

The effect of vaccination on incidence of FDPMET is present in Table 7.When the vaccines were evaluated separately, the incidence of FDPMETtended to be different among the treatments (P-value=0.064).Additionally, when the vaccines were evaluated grouped as subcutaneousor intravaginal vaccines, the subcutaneous vaccines were associated witha significantly lower odds of FDPMET (P-value=0.047).

TABLE 7 Effects of different vaccine formulations on incidence of farmdiagnosed puerperal metritis. Vaccines were evaluated separately inModel 1, and grouped in Model 2. Age in days and body condition score atenrollment were offered to both models Puerperal metritis Model andincidence Coefficients variables (%) (SE) Odds ratio (95% CI) P-valueModel 1 Control 27.62 Ref. baseline 0.064 Vaccine 1 11.11 −0.73 (0.38)0.33 (0.13-0.85) Vaccine 2 16.98 −0.24 (0.33) 0.54 (0.23-1.23) Vaccine 320.75 0.005 (0.31) 0.69 (0.31-1.51) Vaccine 4 33.96   0.68 (0.27) 1.35(0.66-2.74) Vaccine 5 19.23 −0.09 (0.32) 0.62 (0.28-1.40) Intercept−1.34 (0.14) Model 2 Control 27.62 Ref. baseline 0.047 Subcutaneous16.25 −0.43 (0.18) 0.51 (0.28-0.93) Intravaginal 26.67   0.19 (0.18)0.95 (0.52-1.75) Intercept −1.20 (0.12)

The effect of vaccination on rectal temperature at 6±1 DIM is presentedin FIG. 3. Rectal temperature was not statistically different among thetreatment groups when the vaccines were evaluated separately(P-value=0.14). However, rectal temperature was statistically differentbetween the treatment groups when the vaccines were evaluated grouped ascontrol, subcutaneous vaccines or intravaginal vaccines (P-value=0.018).Subcutaneous vaccination was associated with a significant reduction inrectal temperature at 6±1 DIM.

Effect of vaccination on incidence of endometritis and uterine secretionculture outcomes. Vaccines were not effective in preventingendometritis, when evaluated separately or when grouped as subcutaneousand intravaginal vaccines (P-value=0.99). Endometritis incidence was8.57%, 7.89%, 12.12%, 7.50%, 9.09%, and 9.76% for control, vaccine 1,vaccine 2, vaccine 3, vaccine 4, and vaccine 5, respectively. Theincidence of endometritis was 9.01% and 9.46% for subcutaneous andintravaginal vaccines, respectively. Additionally, there was nosignificant effect of vaccination on the likelihood of intrauterinebacterial contamination (Table 8).

TABLE 8 Effects of different vaccine formulations on incidence ofintrauterine Escherichia coli at 2 ± 1 DIM, Fusobacterium necrophorum at6 ± 1 DIM and Trueperella pyogenes at 35 ± 3 DIM. Vaccines wereevaluated separately in Model 1, Model 3 and Model 5; and grouped inModel 2, Model 4 and Model 6. Age in days and body condition score atenrollment were offered to all models Model and Cows positive forCoefficients Odds ratio (95% variables intrauterine culture (%) (SE) CI)P-value Model 1 E. coli Control 55.00 Ref. baseline 0.63 Vaccine 1 47.06−0.02 (0.25) 0.75 (0.38-1.48) Vaccine 2 46.15 −0.06 (0.25) 0.72(0.36-1.41) Vaccine 3 40.38 −0.35 (0.26) 0.54 (0.27-1.06) Vaccine 450.94   0.12 (0.25) 0.86 (0.44-1.68) Vaccine 5 50.00   0.04 (0.26) 0.80(0.40-1.58) Intercept   1.96 (1.13) Model 2 E. coli Control 55.00 Ref.baseline 0.26 Subcutaneous 44.52 −0.21 (0.14) 0.66 (0.40-1.10)Intravaginal 50.49   0.01 (0.16) 0.83 (0.48-1.44) Intercept Model 3 F.necrophorum Control 48.98 Ref. baseline 0.76 Vaccine 1 36.00 −0.39(0.26) 0.59 (0.29-1.18) Vaccine 2 48.00   0.11 (0.26) 0.96 (0.49-1.90)Vaccine 3 48.00   0.11 (0.26) 0.96 (0.49-1.90) Vaccine 4 47.17   0.07(0.25) 0.93 (0.48-1.81) Vaccine 5 44.00 −0.05 (0.26) 0.82 (0.41-1.62)Intercept −0.19 (0.11) Model 4 F. necrophorum Control 48.98 Ref.baseline 0.74 Subcutaneous 44.00 −0.09 (0.14) 0.82 (0.49-1.36)Intravaginal 45.63 −0.02 (0.16) 0.87 (0.50-1.52) Intercept −0.15 (0.11)Model 5 T. pyogenes Control 14.49 Ref. baseline 0.37 Vaccine 1  5.26−0.80 (0.63) 0.30 (0.06-1.46) Vaccine 2 21.21   0.77 (0.42) 1.44(0.48-4.32) Vaccine 3 12.50   0.05 (0.46) 0.70 (0.21-2.30) Vaccine 412.12   0.06 (0.50) 0.70 (0.20-2.52) Vaccine 5  7.32 −0.49 (0.54) 0.41(0.10-1.61) Intercept −16.55 (5.48)  Model 6 T. pyogenes Control 14.49Ref. baseline 0.50 Subcutaneous 12.61   0.01 (0.26) 0.74 (0.30-1.82)Intravaginal  9.46 −0.32 (0.31) 0.53 (0.19-1.53) Intercept −16.66(5.48) 

Effect of vaccination on reproduction. Cows that received subcutaneousvaccination were 1.36 times more likely to conceive when compared tocontrol cows (P-value=0.04). However, for cows that receivedintravaginal vaccines, the likelihood of conceiving was notstatistically different from control cows (Hazard ratio=1.12,P-value=0.46). Age in days at enrollment and BCS at enrollment wereretained in the model for this analysis (P-value=0.02 and 0.01,respectively). The improvement in reproductive performance is furtherillustrated by survival analysis (FIG. 5) which demonstrates that cowsvaccinated with subcutaneous vaccines became pregnant significantlyfaster than control cows and cows that received intravaginalvaccination.

Serological responses to vaccination. The effect of vaccination onELISA-detected serum IgG against several antigens is presented in FIG.4. In general, vaccination induced a significant increase in serum IgGtiters against all antigens; subcutaneous vaccination was more effectiveat increasing serum IgG titers than intravaginal vaccination.

FIG. 5 shows an example of Kaplan-Meier survival analysis ofcalving-to-conception interval by treatment grouped as control,subcutaneous vaccines and intravaginal vaccines. The vaccinationadministrations were: control (no vaccine), subcutaneous vaccines(vaccines 1, 2, and 3 combined) and intravaginal vaccine (vaccines 4 and5 combined). The Y axis is the % of cows not pregnant and the X axis isthe days from parturition until pregnancy. The mediancalving-to-conception interval for subcutaneous vaccines (innerinterrupted line), intravaginal vaccines (middle interrupted line), andcontrol (solid line) was 94, 114, and 120 respectively. (P-value=0.04).The slope of the line representing the subcutaneous vaccine is steepestand demonstrates that an increased number of cows became pregnant in thesubcutaneous vaccine group when compared to the intravaginal vaccine andcontrol groups.

As will be apparent from the foregoing results presented in thisExample, the effects of 5 different vaccine formulations (3 subcutaneousvaccines and 2 intravaginal vaccines) containing different combinationsof proteins (FimH, F. necrophorum leukotoxin (LKT), T. pyogenes pyolysin(PLO) and inactivated whole cells (E. coli, F. necrophorum and T.pyogenes) on the uterine health of dairy cows are different in thatsubcutaneous vaccination significantly decreased the incidence ofpuerperal metritis, whereas intravaginal vaccination was not effective.

Puerperal metritis is characterized by inflammation of the entirethickness of the uterine walls, and is associated with signs of systemicillness such as dullness, decreased milk yield and fever. When diagnosedas described herein, puerperal metritis incidence was 12.12% and whendiagnosed by farm workers it was 27.62%. This discrepancy can beattributed to the period during which the cows were monitored; whereasfarm workers monitored the cows daily during their first 20 days afterparturition, the research team examined the cows at 6±1 days aftercalving. Cows were examined at this time point because metritis peaks inthe first 7 days after calving. However, in general, the effect ofvaccination on puerperal metritis was consistent between the researchgroup's and the farm workers' diagnoses; subcutaneous vaccinationsignificantly lowered the incidence of puerperal metritis, whereasintravaginal vaccine was not effective in preventing the disease.

E. coli and F. necrophorum are gram-negative bacteria, characterized bythe presence of lipopolysaccharide (LPS) in their outer membrane, andare known etiological agents of puerperal metritis; LPS is known tocause increased body temperature in cattle. Although vaccination did notsignificantly decrease the percentage of cows that were positive forintrauterine E. coli and F. necrophorum, subcutaneously vaccinated cowsdid have a lower rectal temperature at 6±1 DIM. This suggests that, evenin the presence of bacteria in the uterus, immunized cows were lesslikely to develop systemic signs caused by LPS released from E. coli andF. necrophorum. It is known that reducing the bacterial load of E. colidecreases the severity of the disease; therefore, immunization decreasedthe pathogen-load inside the uterus.

Mucosal immune responses can be effectively induced by theadministration of vaccines onto mucosal surfaces, whereas subcutaneousand intramuscular vaccines typically fail to induce mucosal immunity,and are less effective in preventing infection of mucosal surfaces.Promising results regarding prevention of human UTI by intravaginalimmunization with a whole-cell vaccine have already been reported.However, it is not known how local synthesis of specific antibodies byuterine antibody-secreting cells contributes to uterine immunity. In thepresent study, intravaginal immunization was not effective in preventinguterine diseases.

In general, subcutaneous vaccination increased the serum levels of IgGagainst E. coli, FimH, F. necrophorum, LKT, T. pyogenes, and PLO.

It is known that F. necrophorum LKT is highly toxic to bovine PMNs,inducing apoptosis-mediated killing of them; this toxicity isdose-dependent. It is possible that immunizing the cows against LKTmight have reduced the detrimental effect of this toxin on intrauterinePMNs, improving the ability of the innate immune system to eliminatebacterial infections from the uterus through phagocytosis. RecruitedPMNs are key players in the immune defense of the uterus; reducedmigration of PMNs 2 weeks before calving is associated with retainedplacenta, and lower phagocytic activity and oxidative burst capacity ofPMNs are associated with occurrence of metritis and endometritis.

The incidence of puerperal metritis was significantly decreased withprepartum subcutaneous vaccination with vaccines containing differentcombinations of proteins (FimH, LKT, PLO) and inactivated whole cells(E. coli, F. necrophorum and T pyogenes). In contrast, intravaginalvaccination was not effective in decreasing the incidence of puerperalmetritis. Thus a commercially produced vaccine against metritis couldbecome an integral part of a preventive strategy against metritis, whichwould be expected to reduce incidence of the disease and reduce use ofantibiotics thereby alleviating both animal distress and the overallnegative economic impact of metritis on the dairy industry.

While the disclosure has been particularly shown and described withreference to specific embodiments (some of which are preferredembodiments), it should be understood by those having skill in the artthat various changes in form and detail may be made therein withoutdeparting from the spirit and scope of the present disclosure asdisclosed herein.

What is claimed is:
 1. A method of improving reproductive function of aruminant comprising subcutaneously administering to the ruminant aveterinary composition comprising inactivated whole cells of Escherichiacoli (E. coli), Trueperella pyogenes (T. pyogenes), Fusobacteriumnecrophorum (F. necrophorum), and F. necrophorum leukotoxin (LKT)protein, E. coli type 1 fimbrial adhesin (FimH) protein, and T. pyogenespyolysin (PLO) protein, such that the reproductive function of theruminant is improved, wherein the improving the reproductive function ofthe ruminant comprises reducing a calving-to-conception intervalrelative to a calving-to-conception interval of a control, wherein thecontrol is a calving-to-conception interval from a ruminant thatreceived an intravaginal administration of the veterinary composition.2. The method of claim 1, wherein the ruminant is a cow.